Our laboratory has a long-standing interest in non-Pgp mediated mechanisms of drug resistance, having established several cell line models of resistance focusing on the ABC half-transporter ABCG2. We successfully cloned ABCG2 from a mitoxantrone-resistant colon cancer cell line, S1-M1-80, that exhibited an ATP-dependent reduction in drug accumulation. Comprising 6 transmembrane domains and a single ATP binding domain, the gene encodes a half-transporter molecule, and it is thought that dimerization is required for activity. Overexpression of ABCG2 renders cells resistant to mitoxantrone and to the camptothecins, topotecan and SN-38 (the active metabolite of irinotecan). Both substrates and inhibitors of ABCG2 have been discovered at an accelerating pace, and the variation in inhibitors rivals that described for P-glycoprotein. There is increasing evidence supporting a role for ABCG2 in oral absorption of pharmacologic agents. We have worked on structure and function relationships in the protein. We identified a naturally occurring mutation in ABCG2 (R482T; R482G) that alters substrate and inhibitor specificity; and then carried out a sequence analysis of ABCG2, identifying single nucleotide polymorphisms. We and others identified impaired transport was observed in cells bearing the Q141K. Since gastrointestinal absorption of topotecan has been related to ABCG2 expression in the intestinal epithelium, a clear implication of this work is that the Q141K SNP could be associated with increased exposure to substrate drugs. To the extent that ABCG2 is involved in drug excretion, this SNP could increase exposure to substrates such as imatinib, irinotecan or topotecan. In an attempt to identify the mechanism of dimerization of ABCG2, our laboratory has studied a GXXXG dimerization motif in the transmembrane helix 1, finding that while it is critical for normal transport activity, normal cross-linking is retained in cells expressing glycine to leucine mutations at amino acids 406 and 410. We recently discovered that when a nearby residue, 402, was also mutated, that the protein was completely destabilized. This despite that mutation of the residue at amino acid 402 has no impact alone. In collaboration with Balasz Sarkadi, we have generated Sf9 insect vectors to allow transfection in the high expression insect system. This system is more tolerant of misfolded protein and has already been shown to generate functional ABCG2 molecules. This system will allow co-immunoprecipitation studies that are needed to provide confirmation of the impact of our mutations on dimerization. More recently, additional residues highly conserved or predicted to be important in the model generated by Di Xia are being evaluated. Mutation of the highly conserved amino acid residue 553 results in loss of protein expression on the mammalian cell surface, and expression of a nonfunctional protein on the insect cell surface. One goal has been to develop a specific, functional assay for ABCG2. Since mitoxantrone is also a substrate for P-glycoprotein, we were interested in pheophorbide a when it was first described by Shinkel et al as the agent that produced phototoxicity in ABCG2 knockout mice. We reasoned that since phototoxicity had not been observed in the intensively studied Pgp knockout mice, pheophorbide a might be an ABCG2-specific substrate. This could allow more accurate clinical detection of ABCG2. We tested pheophorbide a selected cell lines and in the HEK 293 clones transfected with pcDNA vectors encoding ABCG2 with wild type, mutant and SNP sequences and found a tight correlation between cell surface expression as measured by 5D3 antibody and pheophorbide a efflux as measured by inhibition with FTC. No transport was observed in cells expressing Pgp or MRP. Hoechst 33342 DNA stain has also been described as a substrate for ABCG2.